PROJECT SUMMARY Traumatic Spinal Cord Injury (SCI) is a devastating condition currently affecting approximately 300,000 people in the United States. The prevalence of severe chronic pain in SCI is 16%-63%. Following SCI, rapid activation of microglia at the injury site likely contributes to establishment of chronic pain through the release of pro- inflammatory signaling mediators. VGF (nonacronymic) is a neuropeptide precursor that is upregulated upon neuronal injury, and VGF expression is rapidly increased after contusion SCI. Dr. Vulchanova's laboratory was the first to show that VGF-derived peptides signal to microglia, and established that the VGF-derived peptide TLQP-21 contributes to both the development and maintenance of hypersensitivity after peripheral nerve injury and inflammation. TLQP-21 activates the Complement 3a (C3aR1) receptor. Because C3aR1 is upregulated in microglia following CNS injury, I hypothesize that C3aR1-expressing microglia are the cellular target of TLQP-21. This idea is supported by my preliminary results in microglial cultures indicating that TLQP-21 evokes Ca2+ transients in microglia. Signaling via C3aR1 is known to activate the mitogen-activated protein kinase family, including ERK. Activated ERK is increased in microglia after SCI and is associated with hypersensitivity by mediating release of PGE2. My overall hypothesis is that TLQP-21 and C3aR1 increase spinal microglial ERK and PGE2 signaling, ultimately leading to SCI neuropathic pain. Specific aim 1: Characterize behavioral and spinal microglial correlates of neuropathic pain after SCI and evaluate the involvement of TLQP-21 and C3aR1 in SCI pain behavior. I will evaluate behavioral signs of hypersensitivity and spontaneous pain and will quantify Iba1, pERK, TLQP-21 and C3aR1 using immunohistochemistry and in situ hybridization. Additionally I will examine the contribution of TLQP-21 and C3aR1 to the maintenance of pain behavior following SCI. My working hypothesis is that disruption of TLQP-21 and C3aR1 signaling will attenuate the maintenance of pain-related behaviors after SCI. I will use TLQP-21 sequestration as well as a C3aR1 antagonist to examine the function of TLQP-21 and C3aR1 in the establishment of pain behaviors following SCI. Specific aim 2: Establish the function of TLQP-21 and C3aR1 on spinal microglia in our SCI model. I will use Ca2+ imaging in the adult spinal cord slice to test the working hypothesis that TLQP-21-evoked C3aR1-dependent calcium transients are potentiated in microglia after SCI. Additionally, utilizing an ELISA, I will examine PGE2 release from injured spinal cord slices to test the working hypothesis that TLQP-21 evoked PGE2 release is potentiated following SCI. These studies will have a significant impact because they represent the first examination of the function of the VGF-derived peptide TLQP-21 on spinal microglial signaling and on chronic pain following SCI. The outcomes of the proposed project have the potential to impact the identification of TLQP-21 and C3aR1 as novel therapeutic targets for SCI-induced neuropathic pain.